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. 2007 Mar 12;176(6):795–805. doi: 10.1083/jcb.200701066

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Copyright © 2007, The Rockefeller University Press

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Figure 7. — Microtubule attachment in mitosis is not required for CENP-A assembly at centromeres. (A) Schematic of cell synchronization, transfection, and labeling protocol. (B) Representative image of cells transiently transfected with a transcription-mediated BubR1 RNAi gene during late S/G2 phase, after which cells were arrested again with thymidine. After quenching CENP-A–SNAP with BG, thymidine inhibition was released, followed by addition of nocodazole to block spindle microtubule assembly. Newly made CENP-A–SNAP was pulse labeled with TMR-Star in the subsequent S/G2 (8 h after release from thymidine). 15 h after thymidine release, two of three cells (1 and 2) were premitotic (based on the centromere number consistent with unresolved sister centromere pairs) and had not loaded CENP-A–SNAP, whereas the third cell had exited mitosis without chromosome segregation and cytokinesis (centromere number consistent with an 8N DNA content in which sister centromeres are resolved) and had loaded CENP-A–SNAP. Centromere number per cell is indicated in the HA image. (C) Percentages of cells that had loaded CENP-A after the manipulations in A with the siRNAs as indicated. Bracketed numbers represent number of cells counted for each condition. (D) Frequency distribution of centromere numbers for cells with (TMR positive; red) or without (TMR negative; blue) CENP-A loading.